The goal of the project consists in developing a new strategy of cutting genomic DNA, which converts the most common restriction enzymes into rare and super-rare cutters yielding DNA fragments in the range 0.1-10 Mbp. The major tool for reaching the goal is Peptide Nucleic Acid (PNA), a synthetic mimic of oligonucleotides, which carries DNA bases attached to a polyamide backbone. Homopyrimidine PNAs form a very strong and highly sequence-specific complex with duplex DNA. In this complex, called P-loop, two homopyrimidine PNA molecules form a triplex with the complementary purine strand leaving the pyrimidine DNA strand displaced. A major idea underlying the project is that sequence-specific PNA binding to relatively short DNA sequences blocks DNA recognition by methyltransferase (methylase) if the PNA binding site overlaps with the methylase binding site. If this were true and PNA binding were sufficiently sequence-specific, it would open the possibility of applying PNA for the cleavage of genomic DNA: after methylation of a PNA/DNA complex, PNA is removed and DNA is cleaved by an appropriate restriction enzyme only at the sites that were protected against methylation by PNA. To reach the objectives of the project, the PNA/DNA complex formation for various PNA modifications will be studied in great detail. The kinetics and mechanism of interaction with duplex DNA of bis-PNA, consisting of two PNA molecules connected by a flexible linker/bis-PNA carrying additional positive charges and other PNA modifications, will be studied. Based on the detailed understanding of the mechanism of the P-loop formation, the most appropriate PNA modification will be chosen for genome cutting. The mechanism of interaction of methylases with PNA/DNA complex will be studied to select conditions under which PNA prevents methylation of the sites protected due to PNA binding. A hypothesis will be tested that methylase significantly enhances the selectivity of discrimination between perfect and imperfect PNA binding because imperfect PNA/DNA complexes do not prevent methylation. Different PNA constructions will be tested on several genomic DNAs in combination with different methylation-restriction enzymatic pairs. The major result of the project will consist in the development of a new huge class of genome cutters, which selectively cut genomic DNAs in various ranges of lengths, from one per 100 kbp to one per 1 Mbp and even rarer, if necessary. This is the range where very few cutters, if any, are presently available. This could significantly simplify numerous techniques in the entire field of DNA analysis, including genome mapping, cloning and sequencing.